Mechanisms of Hemostatic Protease Inhibition by Serpins

Summary

Principal Investigator: Ingrid Verhamme
Abstract: The long-term goal is to define the molecular mechanisms of thrombin (T) inhibition by the serpins, heparin cofactor II (HCII) and plasminogen activator inhibitor-1 (PAI-1), implicated in arterial thrombosis. Thrombin localized on fibrin (Fbn) and the glycosaminoglycans (GAGs) dermatan sulfate (DS)and heparin, reacts with HCII and platelet PAI-1, in GAG-accelerated mechanisms different from thrombin inhibition by antithrombin (AT), accelerated by high affinity heparin, present only in trace amounts. Unlike AT, HCII is a unique inhibitor of arterial thrombosis, as only HCII in the presence of DS is capable of inhibiting Fbn-bound thrombin. Thrombin exosites I and II are hypothesized to play different roles in these processes. Exosite I binds HCII and PAI-1 directly, whereas exosite II - heparin binding may modulate HCII and PAI-1 turnover. These steps are absent in the T - AT reaction. DS bound outside exosite II is hypothesized to act as template for inhibition by HCII of exosite ll-blocked thrombin, meizothrombin (MzT), and MzT(desFI). The identity of this site;the HCII and PAI-1 substrate pathways;the mechanisms of DS- selective inhibition of Fbn-bound thrombin by HCII, and of fibrinogen (Fbg) and Fbn regulation of thrombin inhibition by PAI-1 are all unknown. The studies will resolve these significant gaps, by using fluorescence equilibrium binding, steady-state and rapid kinetics with native thrombin, HCII and PAI-1, and specific loss- of-function mutants. They will test the hypotheses: that the exosite roles in the GAG-catalyzed thrombin inactivation mechanisms by HCII, PAI-1 and AT are distinctly different;that GAG binding outside exosite II on thrombin mediates inhibition by HCII;and that Fbg and Fbn regulate thrombin inhibition by these serpins differentially. Specific aims are: (1) To quantitate binding and chemical steps in the sequence of molecular events in the GAG-catalyzed thrombin inactivation and substrate pathways of HCII and PAI-1, compared to AT;(2) Tocharacterize the DS-binding site outside exosite II in thrombin and MzT, and its role in thrombin and MzT inhibition;and (3) To determine the contributions of Fbg and Fbn binding to exosite I and GAGs in thrombin protection from HCII, PAI-1, and AT. These mechanism-based studies are relevant to understanding the selective, localized regulation of thrombin activity by HCII and PAI-1, and serpin turnover, in arterial clots. They may facilitate development of novel anticoagulants based on HCII and DS specifically targeted to arterial thrombosis.
Funding Period: ----------------2006 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Exosites in the substrate specificity of blood coagulation reactions
    P E Bock
    Department of Pathology, Vanderbilt University, Nashville, TN 37232 2561, USA
    J Thromb Haemost 5:81-94. 2007
  2. pmc The dimeric structure of factor XI and zymogen activation
    Yipeng Geng
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN, USA
    Blood 121:3962-9. 2013
  3. pmc A sequential mechanism for exosite-mediated factor IX activation by factor XIa
    Yipeng Geng
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA
    J Biol Chem 287:38200-9. 2012
  4. pmc Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa
    Y Geng
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA
    J Thromb Haemost 11:1374-84. 2013
  5. pmc Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation
    Ingrid M Verhamme
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 283:26137-47. 2008
  6. pmc Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation
    Stephen B Smith
    Department of Pathology, Vanderbilt University, Nashville, Tennessee 37232, USA
    J Biol Chem 283:6696-705. 2008
  7. pmc Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli
    Suryakala Sarilla
    Department of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232, USA
    Anal Biochem 406:166-75. 2010
  8. pmc Sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor II
    Suryakala Sarilla
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 285:8278-89. 2010
  9. pmc Activation of factor XI by products of prothrombin activation
    Anton Matafonov
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232 6305, USA
    Blood 118:437-45. 2011
  10. pmc Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex
    Ingrid M Verhamme
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Anal Biochem 421:489-98. 2012

Scientific Experts

  • Ingrid Verhamme
  • David Gailani
  • Suryakala Sarilla
  • Yipeng Geng
  • Anton Matafonov
  • Mao Fu Sun
  • Stephen B Smith
  • Y Geng
  • Lawrence C Thompson
  • Sally Y Habib
  • P E Bock
  • D Gailani
  • Stephanie A Smith
  • S P Bajaj
  • J Emsley
  • James H Morrissey
  • Qiufang Cheng
  • M F Sun
  • S Paul Bajaj
  • Amanda Messer
  • Duane E Day
  • Cynthia B Peterson
  • David S Ginsberg
  • John P Sheehan
  • Sumit Goswami
  • Vladimir Serebrov
  • David B Friedman
  • Dmitri V Kravtsov
  • Diana R Arnett
  • Douglas M Tollefsen
  • Paul E Bock
  • P Panizzi

Detail Information

Publications11

  1. pmc Exosites in the substrate specificity of blood coagulation reactions
    P E Bock
    Department of Pathology, Vanderbilt University, Nashville, TN 37232 2561, USA
    J Thromb Haemost 5:81-94. 2007
    ..The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis...
  2. pmc The dimeric structure of factor XI and zymogen activation
    Yipeng Geng
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN, USA
    Blood 121:3962-9. 2013
    ..For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit...
  3. pmc A sequential mechanism for exosite-mediated factor IX activation by factor XIa
    Yipeng Geng
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee 37232, USA
    J Biol Chem 287:38200-9. 2012
    ..Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain...
  4. pmc Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa
    Y Geng
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA
    J Thromb Haemost 11:1374-84. 2013
    ..A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an exosite required for binding of FIX to activated FXI (FXIa)...
  5. pmc Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation
    Ingrid M Verhamme
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 283:26137-47. 2008
    ....
  6. pmc Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation
    Stephen B Smith
    Department of Pathology, Vanderbilt University, Nashville, Tennessee 37232, USA
    J Biol Chem 283:6696-705. 2008
    ..The results indicate that two forms of activated factor XI are generated during coagulation, and that each half of a factor XIa dimer behaves as an independent enzyme with respect to factor IX...
  7. pmc Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli
    Suryakala Sarilla
    Department of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232, USA
    Anal Biochem 406:166-75. 2010
    ..This weaker binding may be attributed to interference of the Asn(169)N-glycan with the HCII heparin-binding site...
  8. pmc Sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor II
    Suryakala Sarilla
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
    J Biol Chem 285:8278-89. 2010
    ..The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation...
  9. pmc Activation of factor XI by products of prothrombin activation
    Anton Matafonov
    Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232 6305, USA
    Blood 118:437-45. 2011
    ....
  10. pmc Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex
    Ingrid M Verhamme
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
    Anal Biochem 421:489-98. 2012
    ....
  11. pmc Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition
    Lawrence C Thompson
    Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA
    Protein Sci 20:353-65. 2011
    ..Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability...